Novel serine protease BSSP4

ABSTRACT

There are provided proteins having the amino acid sequences represented by SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20; proteins having amino acid sequences derived from these amino acid sequences by deletion, substitution or addition of one to several amino acids; and nucleotide sequences encoding the same; transgenic non-human animals with altered expression level of a serine protease BSSP4; an antibody against BSSP4; and a method for detecting BSSP4 in a specimen by using the antibody.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of application Ser. No. 09/856,298, which is a 371national stage application of PCT/JP99/06472, filed Nov. 19, 1999, theentire contents of both applications being incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to isolated polynucleotides of human andmouse serine proteases (hereinafter referred to as “hBSSP4” and“mBSSP4”, respectively, and, in case no differentiation thereof fromeach other is needed, simply referred to as “BSSP4”), and theirhomologous forms, mature forms, precursors and polymorphic variants aswell as a method for detecting thereof. Further, it relates to hBSSP4and mBSSP4 proteins, compositions containing hBSSP4 and mBSSP4polynucleotides and proteins, as well as their production and use.

BACKGROUND OF THE INVENTION

In general, proteases are biosynthesized as inactive precursors. Theyundergo limited hydrolysis in molecules to convert into activated typeproteases. In so far as enzymes are proteases, they have an activity forhydrolyzing a peptide bond, while their action modes are variedaccording to kinds of proteases. According to a particular kind ofcatalytic site, proteases are divided into serine proteases, cysteineproteases, aspartate proteases, metal proteases and the like. Proteasesof each kind have a variety of properties, ranging from a proteasehaving general digestive properties to a protease having variousregulatory domains and strict substrate specificity, therebyspecifically hydrolyzing only characteristic proteins.

Further, proteins undergo various processing even after translation toproduce active proteins. In many secretory proteins, a protein are firstsynthesized on the ribosome in cytoplasm as an inactive precursor(pro-form) which comprises an active protein bearing at the N-terminusthereof a peptide of about 15 to 60 amino acids responsible forsecretion (secretory signal). This peptide region is concerned with themechanism for passing through the cell membrane and is removed uponcleavage by a specific protease during the passage through the membrane,in almost all the cases, to produce a mature protein. A secretory signalhas a broad hydrophobic region comprising hydrophobic amino acids in themiddle of the sequence, and basic amino acid residues at a site close tothe N-terminus. A secretory signal is a synonym of a signal peptide. Inaddition, in some proteins, a peptide moiety which functions as asecretory signal is further attached to the N-terminus of the inactiveprecursor (pro-form). Such a protein is called a prepro-protein(prepro-form).

For example, trypsin is present as a prepro-form immediately aftertranslation into amino acids. After being secreted from cells, it ispresent as a pro-form and is converted into active trypsin in duodenumupon limited hydrolysis by enteropeptidase or by trypsin itself.

The optimal pH range of serine proteases is neutral to weak alkalineand, in general, many of them have a molecular weight of about 30,000 orlower. All proteases of blood coagulation, fibrinolysis and complementsystems having a large molecular weight belong to trypsin-like serineproteases. They have many regulator domains and form a protease cascadewhich is of very importance to reactions in a living body.

Recently, cDNAs and amino acid sequences of many novel proteases havebeen determined by PCR for consensus sequences of serine proteases usingoligonucleotide primers. According to this method, novel proteases havebeen found by various researchers such as Yamamura et al. (Yamanura, Yet al., Biochem. Biophys. Res. Commun., 239, 386, 1997), Gschwend, etal. (Gschwend, T. P. et al., Mol. Cell. Neurosci., 9. 207, 1997), Chenet al. (Chen, Z-L, et al., J. Neurosci., 15, 5088, 1995) and others.

SEQ ID NO: 3 of JP 9-149790 A discloses neurosin as a novel serineprotease. Neurosin has also been reported in Biochimica et ByophysicaActa, 1350, 11-14, 1997. By this, there is provided a method for massproduction of neurosin using the serine protease gene and a method forscreening specific inhibitors using the enzyme. In addition, thescreening method has been shown to be useful for screening medicines fortreating various diseases.

Serine proteases expressed in a brain-nerve system such as neurosin areconsidered to play various roles in the brain-nerve system. Therefore,there is a possibility that isolation of a gene encoding a novelprotease expressed in a brain-nerve system and production of a proteinusing the gene would be useful for diagnosis or treatment of variousdiseases related to the brain-nerve system.

Nowadays, in general, clinical diagnosis of Alzheimer's disease isconducted based on the diagnosis standard of DSM-IIIR and NINCDS-ADRDA(Mckhann, G. et al., Neurology, 34. 939, 1994) or the diagnosis standardof DSM-IV (American Psychiatric Association; Diagnostic and statisticalmanuals of mental disorders, 4th ed., Washington D.C., AmericanPsychiatric Association, 1994). However, these standards are conditionedby decline of recognition functions which causes a severe disability ina daily life or a social life. Then, it is pointed out that thediagnosis is less scientific objectivity because the diagnosis may beinfluenced by the level of an individual's social life and further thespecialty and experience of a physician who diagnoses particularconditions. In addition, definite diagnosis of Alzheimer's disease isconducted by pathohistological analyses and, in this respect,substantial inconsistency between clinical diagnosis and autopsydiagnosis is pointed out.

At present, image diagnosis is employed as a supplemental means inclinical diagnosis of Alzheimer's diagnosis and it is possible toanalyze brain functions, for example, decline of metabolism and atrophyin specific sites such as hippocampus, parietal lobe of cerebral cortexand the like which are specific for Alzheimer's disease by PET andSPECT. However, to define Alzheimer's disease based on lowering of ablood flow from parietal lobe to temporal lobe is very dangerous. Inaddition, there is few report showing that MRS testicle useful forpatients with dementia including those of Alzheimer's disease. Further,although CT-MRI image diagnosis is used, a lesion of white matter suchas atrophy of brain, PVL or the like is not specific for Alzheimer typedementia. Since it has been reported that atrophy of brain proceeds asgetting older, the above observation is not necessarily found inAlzheimer type dementia. Furthermore, since an image obtained by MRIvaries according to strength of a magnetic field, performance of anapparatus and imaging conditions, numerical data obtain in differentfacilities cannot be compared with each other except atrophic change. Inaddition, there is a limit to image measurement. Further, enlargement ofventricle can be recognized in vascular dementia cases and there arecases wherein atrophy of hippocampus is observed after ischemia ofbasilar artery.

Under these circumstances, many researchers have requested to developbiological diagnosis markers as a means for providing better precisionand objectivity for clinical diagnosis of Alzheimer's disease. At thesame time, the following important roles in the future will be expected.

1) Objective judgment system of effect of medicaments for treatingAlzheimer's disease.

2) Detection of Alzheimer's disease before a diagnosis standard is met,or disease conditions are manifested.

Further, data obtained in different facilities can be compared with eachother by using the same diagnosis marker. Therefore, development ofbiological diagnosis markers is recognized to be a most important fieldamong fields of Alzheimer's disease studies and its future prospectswill be expected. Approaches to development of biological diagnosismarkers up to now are divided into that based on constitute componentsof characteristic pathological changes of Alzheimer's disease such assenile plaque and neurofibril change, and an approach based on othermeasures. Examples of the former include cerebrospinal fluid tauprotein, Aβ and its precursor, βAPP. Examples of the latter includemydriasis test with cholilytic drug, Apo E and other genes relating toAlzheimer's disease. However, no good results are obtained.

Serine proteases are also considered to play important role in cancercells. The reason why extermination of cancer by surgical treatment ortopical irradiation of radioactive ray is difficult is metastasiscapability of cancer. For spread of solid tumor cells in a body, theyshould loosen their adhesion to original adjacent cells, followed byseparating from an original tissue, passing through other tissues toreach blood vessel or lymph node, entering into the circulatory systemthrough stratum basal and endothelial layer of the vessel, leave fromthe circulatory system at somewhere in the body, and surviving andproliferating in a new environment. While adhesion to adjacent epidermalcells is lost when expression of cadherin which is an intercellularadhesive molecule of epithelium is stopped, to break through tissues isconsidered to depend on proteolytic enzymes which decompose anextracellular matrix.

As enzymes which decompose the matrix, mainly, metal proteases (Rha, S.Y. et al., Breast Cancer Research Treatment, 43, 175, 1997) and serineproteases are known. They cooperate to decompose matrix protein such ascollagen, laminin and fibronectin. Among serine proteases known to beconcerned in decomposition of the matrix, in particular, there isurokinase type plasminogen activator (U-PA). U-PA has a role as atrigger specific for a protein decomposition chain reaction. Its directtarget is plasminogen. It is present in blood abundantly and is aprecursor of an inactive serine protease which accumulates inreconstructed sites of tissues such as injured sites and tumors as wellas inflammatory sites. In addition, as proteases which are concerned inmetastasis and infiltration of cancers, for example, a tissue factor,lysosomal type hydrolase and collagenase have been known.

At present, cancer is the top cause of death in Japan and more than200,000 people are died per year. Then, specific substances which can beused as markers for diagnosis and therapy or prophylaxis of cancer arestudied intensively. Such specific substances are referred to as tumormarkers or tumor marker relating biomarkers. They are utilized in aid ofdiagnosis before treatment of cancer, for presuming carcinogenic organand pathological tissue type, for monitoring effect of treatment, forfinding recurrence early, for presuming prognosis, and the like. Atpresent, tumor markers are essential in clinical analyses. Among them,alpha fetoprotein (AFP) which has high specificity to hepatocellularcarcinoma and yolk sac tumor (Taketa K. et al., Tumour Biol., 9, 110,1988), and carcinoembronic antigen (CEA) are used worldwide. In thefuture, tumor markers will be required more and more, and it is desiredto develop, for example, organ specific markers and tumor cell specificmarkers which are highly reliable serologic diagnosis of cancer. Up tonow, humunglandular kallikrein (hK2) which is a serine proteaseexpressed at human prostatic epithelial cells has been reported as amarker for prostatic cancer. And, hK2 has 78% homology with the sequenceof prostatic specific antigen (PSA) and PSA is also used widely as abiochemical marker of prostatic cancer (Mikolajczyk, S. d. et al.,Prostate, 34, 44, 1998; Pannek, J. et al., Oncology, 11, 1273, 1997;Chu, T. M. et al., Tumour Biology, 18, 123, 1997; Hsieh, M. et al.,Cancer Res., 57, 2651, 1997). Further, hK2 is reported to be useful as amarker for not only prostatic cancer but also stomach cancer (Cho, J. Y.et al. Cancer, 79, 878, 1997). Moreover, CYFRA (CYFRA 21-1) formeasuring cytokeratin 19 fragment in serum is reported to be useful forlung cancer (Sugiyama, Y. et al., Japan J. Cancer Res., 85, 1178, 1994).Gastrin release peptide precursor (ProGRP) is reported to be useful as atumor marker (Yamaguchi, K. et al., Japan, J. Cancer Res., 86, 698,1995).

OBJECTS OF THE INVENTION

Thus, the main object of the present invention is to provide a novelserine protease which can be used for treating or diagnosing variousdiseases such as Alzheimer's disease (AD), epilepsy, cancer,inflammation, sterility, prostate hypertrophy and the like in varioustissues such as brain, lung, prostate, testicle, skeletal muscle, liverand the like, and can be used as an excellent marker instead of thatpresently used.

SUMMARY OF THE INVENTION

Under these circumstances, the present inventors have succeeded incloning of cDNA encoding novel human and mouse serine proteases.

In summary, one feature of the present invention is amino acid sequencesof biological active mature serine proteases hBSSP4 and mBSSP4 as wellas nucleotide sequences encoding the amino acid sequences.

That is, they are the amino acid sequence composed of 268 amino acidsrepresened by the 1st to 268th amino acids of SEQ ID NO: 2 and anucleotide sequence encoding the amino acid sequence (the 151st to 954thbases of SEQ ID NO: 1). In addition, they include amino acid sequencessubstantially similar to the amino acid sequence and nucleotidesequences encoding such similar amino acid sequences. Further, theyinclude modified derivatives of proteins having these amino acidsequences. An amino acid sequence substantially similar to a given aminoacid sequence used herein means an amino acid sequence derived from thegiven amino acid sequence by modification such as substitution,deletion, addition and/or insertion of one to several amino acids withmaintaining the same property as that of the protein having the givenamino acid sequence. The modified derivative of the proteins includes,for example, phosphate adduct, sugar chain adduct, metal adduct (e.g.,calcium adduct), the protein fused to another protein such as albuminetc., dimer of the protein, and the like.

Further, they are the amino acid sequence composed of 270 amino acidsrepresened by the 1st to 270th amino acids of SEQ ID NO: 4 and anucleotide sequence encoding the amino acid sequence (the 15th to 960thbases of SEQ ID NO: 3). In addition, they include amino acid sequencessubstantially similar to the amino acid sequence and nucleotidesequences encoding such similar amino acid sequences. Further, theyinclude modified derivatives of proteins having these amino acidsequences.

Further, they are the amino acid sequence composed of 257 amino acidsrepresened by the 1st to 257th amino acids of SEQ ID NO: 6 and anucleotide sequence encoding the amino acid sequence (the 151st to 921stbases of SEQ ID NO: 5). In addition, they include amino acid sequencessubstantially similar to the amino acid sequence and nucleotidesequences encoding such similar amino acid sequences. Further, theyinclude modified derivatives of proteins having these amino acidsequences.

Further, they are the amino acid sequence composed of 97 amino acidsrepresened by the 1st to 97th amino acids of SEQ ID NO: 8 and anucleotide sequence encoding the amino acid sequence (the 151st to 441stbases of SEQ ID NO: 7). In addition, they include amino acid sequencessubstantially similar to the amino acid sequence and nucleotidesequences encoding such similar amino acid sequences. Further, theyinclude modified derivatives of proteins having these amino acidsequences.

Further, they are the amino acid sequence composed of 158 amino acidsrepresened by the 1st to 158th amino acids of SEQ ID NO: 10 and anucleotide sequence encoding the amino acid sequence (the 151st to 624thbases of SEQ ID NO: 9). In addition, they include amino acid sequencessubstantially similar to the amino acid sequence and nucleotidesequences encoding such similar amino acid sequences. Further, theyinclude modified derivatives of proteins having these amino acidsequences.

Further, they are the amino acid sequence composed of 82 amino acidsrepresened by the 1st to 82nd amino acids of SEQ ID NO: 12 and anucleotide sequence encoding the amino acid sequence (the 151st to 396thbases of SEQ ID NO: 11). In addition, they include amino acid sequencessubstantially similar to the amino acid sequence and nucleotidesequences encoding such similar amino acid sequences. Further, theyinclude modified derivatives of proteins having these amino acidsequences.

Further, they are the amino acid sequence composed of 185 amino acidsrepresened by the 1st to 185th amino acids of SEQ ID NO: 14 and anucleotide sequence encoding the amino acid sequence (the 151st to 705thbases of SEQ ID NO: 13). In addition, they include amino acid sequencessubstantially similar to the amino acid sequence and nucleotidesequences encoding such similar amino acid sequences. Further, theyinclude modified derivatives of proteins having these amino acidsequences.

Further, they are the amino acid sequence composed of 80 amino acidsrepresened by the 1st to 80th amino acids of SEQ ID NO: 16 and anucleotide sequence encoding the amino acid sequence (the 151st to 390thbases of SEQ ID NO: 15). In addition, they include amino acid sequencessubstantially similar to the amino acid sequence and nucleotidesequences encoding such similar amino acid sequences. Further, theyinclude modified derivatives of proteins having these amino acidsequences.

Further, they are the amino acid sequence composed of 253 amino acidsrepresened by the 1st to 253th amino acids of SEQ ID NO: 18 and anucleotide sequence encoding the amino acid sequence (the 151st to 909thbases of SEQ ID NO: 17). In addition, they include amino acid sequencessubstantially similar to the amino acid sequence and nucleotidesequences encoding such similar amino acid sequences. Further, theyinclude modified derivatives of proteins having these amino acidsequences.

Further, they are the amino acid sequence composed of 34 amino acidsrepresened by the −49th to −16th amino acids of SEQ ID NO: 2 and anucleotide sequence encoding the amino acid sequence (the 4th to 105thbases of SEQ ID NO: 1). In addition, they include amino acid sequencessubstantially similar to the amino acid sequence and nucleotidesequences encoding such similar amino acid sequences. Further, theyinclude modified derivatives and fragments of proteins having theseamino acid sequences.

Further, they are the amino acid sequence composed of 15 amino acidsrepresened by the −15th to −1st amino acids of SEQ ID NO: 2 and anucleotide sequence encoding the amino acid sequence (the 106th to 150thbases of SEQ ID NO: 1). In addition, they include amino acid sequencessubstantially similar to the amino acid sequence and nucleotidesequences encoding such similar amino acid sequences. Further, theyinclude modified derivatives and fragments of proteins having theseamino acid sequences.

Further, they are the amino acid sequence composed of 259 amino acidsrepresened by the 1st to 259th amino acids of SEQ ID NO: 20 and anucleotide sequence encoding the amino acid sequence (the 227th to1003rd bases of SEQ ID NO: 19). In addition, they include amino acidsequences substantially similar to the amino acid sequence andnucleotide sequences encoding such similar amino acid sequences.Further, they include modified derivatives of proteins having theseamino acid sequences.

Further, they are the amino acid sequence composed of 34 amino acidsrepresened by the −49th to −16th amino acids of SEQ ID NO: 20 and anucleotide sequence encoding the amino acid sequence (the 80th to 181stbases of SEQ ID NO: 19). In addition, they include amino acid sequencessubstantially similar to the amino acid sequence and nucleotidesequences encoding such similar amino acid sequences. Further, theyinclude modified derivatives and fragments of proteins having theseamino acid sequences.

Further, they are the amino acid sequence composed of 15 amino acidsrepresened by the −15th to −1st amino acids of SEQ ID NO: 20 and anucleotide sequence encoding the amino acid sequence (the 182nd to 226thbases of SEQ ID NO: 19). In addition, they include amino acid sequencessubstantially similar to the amino acid sequence and nucleotidesequences encoding such similar amino acid sequences. Further, theyinclude modified derivatives and fragments of proteins having theseamino acid sequences.

Another feature of the present invention is an amino acid sequencecomposed of 317 or 283 amino acids wherein 49 amino acids represented bythe −49th to −1st amino acids or 15 amino acids represented by the −15thto −1st amino acids of SEQ ID NO: 2 are added to the N-terminus side ofthe mature hBSSP4 amino acid sequence represented by SEQ ID NO: 2 (the1st to 268th amino acids) and a nucleotide sequence encoding the aminoacid sequence (the 4th to 954th or 106th to 954th bases of SEQ ID NO:1). In addition, this feature includes amino acid sequencessubstantially similar to the above amino acid sequence and nucleotidesequences encoding these substantially similar amino acid sequences.Further, this feature includes modified derivatives of proteins havingthese amino acid sequences.

Another feature of the present invention is an amino acid sequencecomposed of 319 or 285 amino acids wherein 49 amino acids represented bythe −49th to −1st amino acids or 15 amino acids represented by the −15thto −1st amino acids of SEQ ID NO: 4 are added to the N-terminus side ofthe mature hBSSP4 amino acid sequence represented by SEQ ID NO: 4 (the1st to 270th amino acids) and a nucleotide sequence encoding the aminoacid sequence (the 4th to 960th or 106th to 960th bases of SEQ ID NO:3). In addition, this feature includes amino acid sequencessubstantially similar to the above amino acid sequence and nucleotidesequences encoding these substantially similar amino acid sequences.Further, this feature includes modified derivatives of proteins havingthese amino acid sequences.

Another feature of the present invention is an amino acid sequencecomposed of 306 or 272 amino acids wherein 49 amino acids represented bythe −49th to −1st amino acids or 15 amino acids represented by the −15thto −1st amino acids of SEQ ID NO: 6 are added to the N-terminus side ofthe mature hBSSP4 amino acid sequence represented by SEQ ID NO: 6 (the1st to 257th amino acids) and a nucleotide sequence encoding the aminoacid sequence (the 4th to 921st or 106th to 921st bases of SEQ ID NO:5). In addition, this feature includes amino acid sequencessubstantially similar to the above amino acid sequence and nucleotidesequences encoding these substantially similar amino acid sequences.Further, this feature includes modified derivatives of proteins havingthese amino acid sequences.

Another feature of the present invention is an amino acid sequencecomposed of 308 or 274 amino acids wherein 49 amino acids represented bythe −49th to −1st amino acids or 15 amino acids represented by the −15thto −1st amino acids of SEQ ID NO: 20 are added to the N-terminus side ofthe mature mBSSP4 amino acid sequence represented by SEQ ID NO: 20 (the1st to 259th amino acids) and a nucleotide sequence encoding the aminoacid sequence (the 8th to 1003rd or 182nd to 1003rd bases of SEQ ID NO:19). In addition, this feature includes amino acid sequencessubstantially similar to the above amino acid sequence and nucleotidesequences encoding these substantially similar amino acid sequences.Further, this feature includes modified derivatives of proteins havingthese amino acid sequences.

The present invention also relates to the nucleotide sequencesrepresented by SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 as wellas nucleotide sequences similar to them.

Hereinafter, unless otherwise stated, the nucleotide sequencerepresented by each SEQ ID NO: includes the above-described variousfragments thereof, and similar nucleotide sequences and their fragments.Likewise, the amino acid sequence represented by each SEQ ID NO:includes the above-described various fragments thereof, similarnucleotide sequences and their fragments, and modified derivativesthereof. In addition, unless otherwise stated, BSSP4, hBSSP4, and mBSSP4include proteins having the above-described respective amino acidsequences.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the results of northern blotting using human multipletissue blot membrane.

FIG. 2 illustrates the results of northern blotting using human multipletissue blot II membrane.

FIG. 3 illustrates the results of northern blotting using human multipletissue blot II membrane.

FIG. 4 illustrates the results of northern blotting using mRNA preparedin Example 2 hereinafter.

FIG. 5 illustrates the results of northern blotting using mRNA preparedin Example 2 hereinafter.

FIG. 6 illustrates the plasmid constructed by the method of Example 4hereinafter.

FIG. 7 illustrates the construction of the plasmid by the method ofExample 4 hereinafter.

DETAILED DESCRIPTION OF THE INVENTION

The nucleotide sequences encoding hBSSP4 or mBSSP4 of the presentinvention can be obtained by preparing mRNAs from cells expressing theprotein and converting it into double stranded DNAs according to aconventional manner. For preparing mRNA, guanidineisothiocyanate-calcium chloride method (Chirwin, et al., Biochemistry,18, 5294, 1979) or the like can be used. For preparing poly (A)+RNA fromtotal RNAs, there can be used affinity chromatography using a carrier,for example, Sepharose, latex particles, etc., to which oligo (dT) isattached, and the like. The above-obtained RNA can be used as a templateand treated with reverse transcriptase by using, as a primer, oligo (dT)which is complementary to the poly (A) strand at the 3′-terminus, or arandom primer, or a synthesized oligonucleotide corresponding to a partof the amino acid sequence of hBSSP4 or mBSSP4 to obtain a hybrid mRNAstrand comprising DNA complementary to the mRNA or cDNA. The doublestranded DNA can be obtained by treating the above-obtained hybrid mRNAstrand with E. coli RNase, E. coli DNA polymerase and E. coli DNA ligaseto convert into a DNA strand.

It is also possible to carry out cloning by RT-PCR method using primerssynthesized on the basis of the nucleotide sequence of hBSSP4 or mBSSP4gene and using hBSSP4 or mBSSP4 expressing cell poly (A)+RNA as atemplate. Alternatively, the desired cDNA can be obtained without usingPCR by preparing or synthesizing a probe on the basis of the nucleotidesequence of hBSSP4 or mBSSP4 gene and screening a cDNA library directly.Among genes obtained by these methods, the gene of the present inventioncan be selected by confirming a nucleotide sequence thereof. The gene ofthe present invention can also be prepared according to a conventionalmethod using chemical syntheses of nucleic acids, for example,phosphoamidite method (Mattencci, M. D. et al., J. Am. Chem. Soc., 130,3185, 1981) and the like.

By using the thus-obtained hBSSP4 or mBSSP4 gene, their expression invarious tissues can be examined.

In case of northern blotting analysis, the expression of hBSSP4 isobserved in cerebellum and prostate, and the expression of mBSSP4 isobserved in prostate and skeletal muscle. In case of RT-PCR analysis,the expression of hBSSP4 is observed in brain, placenta and prostate ofhuman fetuses and adults and the expression of mBSSP4 is observed inbrain and placenta of 12-day-old mice. Then, the novel proteases of thepresent invention are presumed to play various roles in brain, prostate,placenta and skeletal muscle. For example, in brain, there is apossibility that they can be used for treatment and diagnosis of braindiseases such as Alzheimer's disease (AD), epilepsy, brain tumor and thelike. Further, in other tissues, there is a possibility that they can beused for treatment and diagnosis of various diseases such as cancer,inflammation, sterility, prostate hypertrophy and the like. Further, itis presumed they may have a certain influence on blood coagulation,fibrinolysis and complement systems.

The human novel serine protease (hBSSP4) is composes of 9 proteins dueto alternative splicing of mRNA.

The protein having the amino acid sequence represented by SEQ ID NO: 2is a human type protein (hBSSP4) and the mature type having serineprotease activity is the polypeptide represented by the 1st to 268thamino acids. As consensus sequences of serine proteases, it hasAla-Ala-His-Cys corresponding to the 39th to 42nd amino acid residues ofSEQ ID NO:2 and Asp-Ser-Gly-Gly-Pro corresponding to the 192nd to 196thamino acid residues of SEQ ID NO:2 and one or more of Asp's are presentbetween the concensus sequences. A nucleotide sequence encoding thisprotein is shown in SEQ ID NO:1.

The protein having the amino acid sequence represented by SEQ ID NO:4 isa human type protein (hBSSP4) and the mature type having serine proteaseactivity is the polypeptide represented by the 1st to 270th amino acids.As consensus sequences of serine proteases, it has Ala-Ala-His-Cyscorresponding to residues of the 39th to 42nd amino acids of SEQ ID NO:4and Asp-Ser-Gly-Gly-Pro corresponding to residues of the represented bythe 192nd to 196th amino acids of SEQ ID NO:1 and one or more of Asp'sare present between the concensus sequences. A nucleotide sequenceencoding this protein is shown in SEQ ID NO:3. This sequence correspondsto SEQ ID NO:1 from which the 943rd to 1217th bases have been removed,and the amino acid sequence represented by SEQ ID NO:4 corresponds tothe amino acid sequence represented by SEQ ID NO:2 in which the 265thamino acid and the subsequent amino acids are different.

The protein having the amino acid sequence represented by SEQ ID NO:6 isa human type protein (hBSSP4) and the mature type having serine proteaseactivity is the polypeptide represented by the 1st to 257th amino acids.As consensus sequences of serine proteases, it has Ala-Ala-His-Cyscorresponding to the 39th to 42nd amino acid residues of SEQ ID NO:6 andAsp-Ser-Gly-Gly-Pro corresponding to the 192nd to 196th amino acidresidues of SEQ ID NO:2 and one or more of Asp's are present between theconcensus sequences. A nucleotide sequence encoding this protein isshown in SEQ ID NO:5. This sequence corresponds to SEQ ID NO:1 fromwhich the 895th to 11208th bases have been removed, and the amino acidsequence represented by SEQ ID NO:6 correspond to the amino acidsequence represented by SEQ ID NO:2 in which the 249th amino acids andthe subsequnent amino acids are different. Further, the nucleotidesequence corresponds to the sequence wherein the 969th to 1036th basesof SEQ ID NO:5 are added to the downstream of the 1282 base of SEQ IDNO:1.

The protein having the amino acid sequence represented by SEQ ID NO: 8is a human type protein (hBSSP4). However, it does not have a consensussequence of serine proteases. Since its expression by mRNA has beenconfirmed, this sequence is consiered to have a certain role. Thenucleotide sequence corresponds to the nucleotide sequence of SEQ ID NO:1 from which the 233rd to 282nd bases have been removed.

The protein having the amino acid sequence represented by SEQ ID NO: 10is a human type protein (hBSSP4). As a consensus sequence of serineproteases, this does not have Ala-Ala-His-Cys corresponding to the 39thto 42nd amino acid residues of SEQ ID NO:10, but has Asp-Ser-Gly-Gly-Procorresponding to residues of the 82nd to 86th amino acids of SEQ IDNO:10. A nucleotide sequence encoding this protein is shown in SEQ IDNO:9. This sequence corresponds to the nucleotide sequence of SEQ IDNO:1 from which the 233rd to 562nd bases have been removed.

The protein having the amino acid sequence represented by SEQ ID NO: 12is a human type protein (hBSSP4). As a consensus sequence of serineproteases, it has Ala-Ala-His-Cys corresponding to residues 39th to 42ndamino acids of SEQ ID NO:12 but does not have Asp-Ser-Gly-Gly-Procorresponding to residues of the 82nd to 86th amino acids of SEQ IDNO:10. A nucleotide sequence encoding this protein is shown in SEQ IDNO:11. This nucleotide sequence corresponds to the nucleotide sequencerepresented by SEQ ID NO:1 from which the 364th to 562nd amino acidshave been removed.

The protein having the amino acid sequence represented by SEQ ID NO:14is a human type protein (hBSSP4). As a consensus sequence of serineproteases, it has Ala-Ala-His-Cys corresponding to the 39th to 42ndamino acid residues of SEQ ID NO:14 but do not have Asp-Ser-Gly-Gly-Procorresponding to residues of the 82^(nd) to 86 ht amino acid of SEQ IDNO:10. A nucleotide sequence encoding this protein is shown in SEQ IDNO:13. This nucleotide sequence corresponds to the nucleotide sequencerepresented by SEQ ID NO:1 from which the 588th to 1145th bases havebeen removed. There is a possibility that the nucleotide sequencerepresented by the 652nd and the subsequent bases of SEQ ID NO: 13 wouldbe “ccc ggg ccc cag cgc ttt tgt gta tat aaa tgt taatgatttt tataggtatttgtaaccctg cccacatatc” SEQ ID NO:49 and the amino acid sequencerepresented by the 168th and the subsequent amino acids of SEQ ID NO: 14would be “Pro Gly Pro Gln Arg Phe Cys Val, Tyr Lys Cys” SEQ ID NO:50.

The protein having the amino acid sequence represented by SEQ ID NO:16is a human type protein (hBSSP4). As a consensus sequence of serineproteases, it has Ala-Ala-His-Cys corresponding to the 39th to 42ndamino acid residues of SEQ ID NO:16 but does not haveAsp-Ser-Gly-Gly-Pro corresponding to the 82nd to 86th amino acidresidues of SEQ ID NO:10. A nucleotide sequence encoding this protein isshown in SEQ ID NO:15. This sequence corresponds to SEQ ID NO: 1 fromwhich the 285th to 562nd bases have been removed.

The protein having the amino acid sequence represented by SEQ ID NO: 18is a human type protein (hBSSP4). As a consensus sequence of serineproteases, it has Ala-Ala-His-Cys corresponding to the 39th to 42ndamino acid residues of SEQ ID NO:18 but does not haveAsp-Ser-Gly-Gly-Pro corresponding to the 82nd to 86th amino acidresidues of SEQ ID NO:10. A nucleotide sequence encoding this protein isshown in SEQ ID NO:17. This sequence corresponds to the sequence whereinthe 721st to 948th bases of SEQ ID NO: 17 is added to the downstream ofthe 720th base of SEQ ID NO: 1, and corresponds SEQ ID NO:1 from whichthe 720th and the subsequent bases have been removed.

The protein having the amino acid sequence represented by SEQ ID NO:20is a mouse type protein (mBSSP4) and the mature type having serineprotease activity is the polypeptide represented by the 1st to 253 aminoacids. As consensus sequences of serine proteases, it hasAla-Ala-His-Cys corresponding to the 39th to 42nd amino acid residues ofSEQ ID NO:20 and Asp-Ser-Gly-Gly-Pro corresponding to the 192nd to 196thamino acid residues of SEQ ID NO:20 and one or more of Asp's are presentbetween the concensus sequences. A nucleotide sequence encoding thisprotein is shown in SEQ ID NO: 19.

The term “pro part” used herein means a part of a pro-form, i.e., thepro-form from which the corresponding active type protein part isremoved. The term “pre part” used herein means a part of a prepro-form,i.e., the prepro-form from which the corresponding pro-form is removed.The term “prepro part” used herein means a part of a prepro-form, i.e.,the prepro-form from which the corresponding active type protein part isremoved.

The amino acid sequence of mature hBSSP4 (the 1st to 268th amino acids)represented by SEQ ID NO: 2 is hBSSP4 mature or active type proteincomposed of 268 amino acids, and the nucleotide sequence encoding theamino acid sequence is composed of 804 bases. The present inventors haveshown that the serine protease activity is maintained even when one toseveral amino acids of the N-terminus of the mature type protein of thepresent invention is deleted or added, while the preferred sequence isthis amino acid sequence. The sequence of the −49th to −1st amino acidsis the prepro or pro part and the amino acid sequence of the −15th to−1st amino acids is the pro part and is considered to be a precursor ofhBSSP4 protein.

The amino acid sequence of mature hBSSP4 (the 1st to 270th amino acids)represented by SEQ ID NO: 4 is hBSSP4 mature or active type proteincomposed of 270 amino acids, and the nucleotide sequence encoding theamino acid sequence is composed of 810 bases. The present inventors haveshown that the serine protease activity is maintained even when one toseveral amino acids of the N-terminus of the mature type protein of thepresent invention is deleted or added, while the preferred sequence isthis amino acid sequence. The sequence of the −49th to −1st amino acidsis the prepro or pro part and the amino acid sequence of the −15th to−1st amino acids is the pro part and is considered to be a precursor ofhBSSP4 protein.

The amino acid sequence of mature hBSSP4 (the 1st to 257th amino acids)represented by SEQ ID NO: 6 is hBSSP4 mature or active type proteincomposed of 257 amino acids, and the nucleotide sequence encoding theamino acid sequence is composed of 771 bases. The present inventors haveshown that the serine protease activity is maintained even when one toseveral amino acids of the N-terminus of the mature type protein of thepresent invention is deleted or added, while the preferred sequence isthis amino acid sequence. The sequence of the −49th to −1st amino acidsis the prepro or pro part and the amino acid sequence of the −15th to−1st amino acids is the pro part and is considered to be a precursor ofhBSSP4 protein.

The amino acid sequence of hBSSP4 (the 1st to 97th amino acids)represented by SEQ ID NO: 8 is a protein composed of 97 amino acids, andthe nucleotide sequence encoding the amino acid sequence is composed of291 bases. The sequence of the −49th to −1st amino acids is the preproor pro part and the amino acid sequence of the −15th to −1st amino acidsis the pro part and is considered to be a precursor of hBSSP4 protein.

The amino acid sequence of hBSSP4 (the 1st to 158th amino acids)represented by SEQ ID NO: 10 is a protein composed of 158 amino acids,and the nucleotide sequence encoding the amino acid sequence is composedof 474 bases. The sequence of the −49th to −1st amino acids is theprepro or pro part and the amino acid sequence of the −15th to −1stamino acids is the pro part and is considered to be a precursor ofhBSSP4 protein.

The amino acid sequence of hBSSP4 (the 1st to 82nd amino acids)represented by SEQ ID NO: 12 is a protein composed of 82 amino acids,and the nucleotide sequence encoding the amino acid sequence is composedof 246 bases. The sequence of the −49th to −1st amino acids is theprepro or pro part and the amino acid sequence of the −15th to −1stamino acids is the pro part and is considered to be a precursor ofhBSSP4 protein.

The amino acid sequence of hBSSP4 (the 1st to 185th amino acids)represented by SEQ ID NO: 14 is a protein composed of 185 amino acids,and the nucleotide sequence encoding the amino acid sequence is composedof 555 bases. The sequence of the −49th to −1st amino acids is theprepro or pro part and the amino acid sequence of the −15th to −1stamino acids is the pro part and is considered to be a precursor ofhBSSP4 protein.

The amino acid sequence of hBSSP4 (the 1st to 80th amino acids)represented by SEQ ID NO: 16 is a protein composed of 80 amino acids,and the nucleotide sequence encoding the amino acid sequence is composedof 240 bases. The sequence of the −49th to −1st amino acids is theprepro or pro part and the amino acid sequence of the −15th to −1stamino acids is the pro part and is considered to be a precursor ofhBSSP4 protein.

The amino acid sequence of hBSSP4 (the 1st to 253th amino acids)represented by SEQ ID NO: 18 is a protein composed of 253 amino acids,and the nucleotide sequence encoding the amino acid sequence is composedof 759 bases. The sequence of the −49th to −1st amino acids is theprepro or pro part and the amino acid sequence of the −15th to −1stamino acids is the pro part and is considered to be a precursor ofhBSSP4 protein.

The amino acid sequence of mature mBSSP4 (the 1st to 259th amino acids)represented by SEQ ID NO: 20 is hBSSP4 mature or active type proteincomposed of 259 amino acids, and the nucleotide sequence encoding theamino acid sequence is composed of 777 bases. The present inventors haveshown that the serine protease activity is maintained even when one toseveral amino acids of the N-terminus of the mature type protein of thepresent invention is deleted or added, while the preferred sequence isthis amino acid sequence. The sequence of the −49th to −1st amino acidsis the prepro or pro part and the amino acid sequence of the −15th to−1st amino acids is the pro part and is considered to be a precursor ofmBSSP4 protein.

In general, many genes of eucaryote exhibit polymorphism and, sometimes,one or more amino acids are substituted by this phenomenon. Further,even in such case, sometimes, a protein maintains its activity. Then,the present invention includes a gene encoding a protein obtained bymodifying a gene encoding any one of the amino acid sequencesrepresented by SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20,artificially, in so far as the protein has the characteristic functionof the gene of the present invention. Further, the present inventionincludes a protein which is a modification of any one of amino acidsequences represented by SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18 and20 in so far as the protein has the characteristics of the presentinvention. Modification is understood to include substitution, deletion,addition and/or insertion. In particular, the present inventors haveshown that, even when several amino acids are added to or deleted fromthe N-terminus amino acid of the hBSSP4 or mBSSP4 mature proteinrepresented by SEQ ID NO: 2, 4, 6 or 20, the resultant sequencemaintains its activity.

That is, the present invention includes a protein comprising any one ofamino acid sequences described in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,16, 18 and 20; an amino acid sequence encoded by any one of nucleotidesequences represented by SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17 and19; or one of these amino acid sequences wherein one to several aminoacids have been substituted, deleted, added and/or inserted, and beingbelonging to serine protease family.

Each codon for the desired amino acid itself has been known and it canbe selected freely. For example, codons can be determined according to aconventional manner by taking into consideration of frequency of use ofcodons in a host to be utilized (Grantham, R. et al., Nucleic AcidsRes., 9, r43, 1989). Therefore, the present invention also includes anucleotide sequence appropriately modified by taking into considerationof degeneracy of a codon. Further, these nucleotide sequences can bemodified by a site directed mutagenesis using a primer composed of asynthetic oligonucleotide encoding the desired modification (Mark, D. F.et al., Proc. Natl. Acad. Sci. USA., 81, 5662, 1984), or the like.

Furthermore, the DNA of the present invention includes DNA which ishybridizable to any one of nucleotide sequences described in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 or nucleotide sequencescomplementary to these nucleotide sequences in so far as the proteinencoded by the nucleotide sequence has the same properties as those ofhBSSP4 or mBSSP4 of the present invention. It is considered that many ofsequences which are hybridizable to a given sequence under stringentconditions have a similar activity to that of a protein encoded by thegiven sequence. The stringent conditions according to the presentinvention includes, for example, incubation in a solution containing5×SSC, 5% Denhardt's solution (0.1% BSA, 0.1% Ficol 1400, 0.1% PVP),0.5% SDS and 20 μg/ml denatured salmon sperm DNA at 37° C. overnight,followed by washing with 2×SSC containing 0.1% SDS at room temperature.Instead of SSC, SSPE can be appropriately used.

Probes for detecting a hBSSP4 or mBSSP4 gene can be designed based onany one of nucleotide sequences described in SEQ ID NOS: 1, 3, 5, 7, 9,11, 13, 15, 17 and 19. Or, primers can be designed for amplifying DNA orRNA containing the nucleotide sequence. To design probes or primers iscarried out routinely by a person skilled in the art. An oligonucleotidehaving a designed nucleotide sequence can be synthesized chemically.And, when a suitable label is added to the oligonucleotide, theresultant oligonucleotide can be utilized in various hybridizationassay. Or, it can be utilized in nucleic acid synthesis reactions suchas PCR. An oligonucleotide to be utilized as a primer has, preferably,at least 10 bases, more preferably 15 to 50 bases in length. Anoligonucleotide to be utilized as a probe has, preferably, 100 bases tofull length.

Moreover, it is possible to obtain a promoter region and an enhancerregion of a hBSSP4 or mBSSP4 gene present in the genome based on thecDNA nucleotide sequence of hBSSP4 or mBSSP4 provided by the presentinvention. Specifically, these control regions can be obtained accordingto the same manner as described in JP 6-181767 A; J. Immunol., 155,2477, 1995; Proc. Natl. Acad. Sci., USA, 92, 3561, 1995 and the like.The promoter region used herein means a DNA region which is presentupstream from a transcription initiation site and controls expression ofa gene. The enhancer region used herein means a DNA region which ispresent in an intron, a 5′-non-translated region or a 3′-non-translatedregion and enhances expression of a gene.

The present invention also relates to a vector comprising the nucleotidesequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19,or a nucleotide sequence encoding the amino acid sequence represented bySEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 ir 20, or a nucleotidesequence similar to these sequences. A nucleotide sequence similar to agive nucleotide sequence used herein means a nucleotide sequence whichis hybridizable to the given nucleotide sequence or its complementarynucleotide sequence under the above-described stringent conditions andencodes a protein having the same properties as those of the proteinencoded by the nucleotide sequence.

The vector is not specifically limited in so far as it can express theprotein of the present invention. Examples thereof include pBAD/His,pRSETA, pcDNA2.1, pTrcHis2A, pYES2, pBlueBac4.5, pcDNA3.1 and pSecTag2manufacture by Invitrogen, pET and PBAC manufactured by Novagen, PGEMmanufactured by Promega, pBluescriptII manufactured by Stratagene, pGEXand pUC18/19 manufactured by Pharmacia, PfastBAC1 manufactured by GIBCOand the like. Preferably, a protein expression vector (described in thespecification of a patent application entitled “Protein expressionvector and its use” and filed by the same applicant on the same day) isused. This expression vector is constructed by using pCRII-TOPO vectordescribed in the Examples hereinafter, or a commercially availableexpression vector, for example pSecTag2A vector or pSecTag2B vector(Invitrogen) and integrating a secretory signal nucleotide sequencesuitable for expression of the protein of the present invention, in the3′ downstream side thereof, a Tag nucleotide sequence, a cleavablenucleotide sequence and a cloning site, into which a nucleotide sequenceencoding a target protein can be inserted, in this order. Morespecifically, it is preferred to use trypsin signal as the secretorysignal, a nucleotide sequence encoding polyhistidine as the Tagnucleotide sequence, and a nucleotide sequence encoding an amino acidsequence which is susceptible to enzyme-specific cleavage, i.e., anucleotide sequence encoding the amino acid sequence ofAsp-Asp-Asp-Asp-Lys SEQ ID NO:51 (said amino acid sequence is recognizedby enterokinase, and the recombinant fusion protein is cleaved at theC-terminus part thereof) as the cleavable nucleotide sequence.

Furthermore, the present invention provides transformed cells having thenucleotide sequence of the present invention in an expressible state bymeans of the above vector. Preferably, host cells to be used for thetransformed cells of the present invention are animal cells and insectcells. However, host cells include any cells (including those ofmicroorganisms) which can express a nucleotide sequence encoding thedesired protein in the expression vector of the present invention andcan secrete extracellularly.

The animal cells and insect cells used herein include cells derived fromhuman being and cells derived from fly or silk worm. For example, thereare CHO cell, COS cell, BHK cell, Vero cell, myeloma cell, HEK293 cell,HeLa cell, Jurkat cell, mouse L cell, mouse C127 cell, mouse FM3A cell,mouse fibroblast, osteoblast, cartilage cell, S2, Sf9, Sf21, High Five™(registered trade mark) cell and the like. The microorganisms usedherein include E. coli and yeast.

The protein of the present invention as such can be expressed as arecombinant fused protein so as to facilitate isolation, purificationand recognition. The recombinant fused protein used herein means aprotein expressed as an adduct wherein a suitable peptide chain areadded to the N-terminus and/or C-terminus of the desired proteinexpressed by a nucleotide sequence encoding the desired protein. Therecombinant protein used herein means that obtained by integrating anucleotide sequence encoding the desired protein in the expressionvector of the present invention and cut off an amino acid sequence whichderived from nucleic acids other than those encoding the desired proteinfrom the expressed recombinant fused protein, and is substantially thesame as the protein of the present invention.

Introduction of the above vector into host cells can be carried out by,for example, transfection according to lipopolyamine method,DEAE-dextran method, Hanahan method, lipofectin method or calciumphosphate method, microinjection, eletroporation and the like.

As described above, the present invention also relates to a process forproducing hBSSP4 of mBSSP4 comprising culturing cells transformed withthe above nucleotide sequence of the present invention and collectingthe produced hBSSP4 of mBSSP4. The culture of cells and separation andpurification of the protein can be carried out by a per se known method.

The present invention also relates to an inhibitor of the novel serineprotease of the present invention. Screening of the inhibitor can becarried out according to a per se known method such as comparing theenzyme activity upon bringing into contact with a candidate compoundwith that without contact with the candidate compound, or the like

The present invention relates to a non-human transgenic animal whoseexpression level of hBSSP4 or mBSSP4 gene has been altered. The hBSSP4or mBSSP4 gene used herein includes cDNA, genomic DNA or synthetic DNAencoding hBSSP4 or mBSSP4. In addition, expression of a gene includesany steps of transcription and translation. The non-human transgenicanimal of the present invention is useful for studies of functions orexpression control of hBSSP4 or mBSSP4, elucidation of mechanisms ofdiseases in which hBSSP4 or mBSSP4 is presumed to be involved, anddevelopment of disease model animals for screening and safety test ofmedicine.

In the present invention, expression of a gene can be modifiedartificially by mutagenizing at a part of several important sites whichcontrol normal gene expression (enhancer, promoter, intron, etc.) suchas deletion, substitution, addition and/or insertion to increase ordecrease an expression level of the gene in comparison with its inherentexpression level. This mutagenesis can be carried out according to aknown method to obtain the transgenic animal.

In a narrow sense, the transgenic animal means an animal wherein aforeign gene is artificially introduced into reproductive cells by generecombinant techniques. In a broad sense, the transgenic animal includesan antisense transgenic animal the function of whose specific gene isinhibited by using antisense RNA, an animal whose specific gene isknocked out by using embryonic stem cells (ES cells), and an animal intowhich point mutation DNA is introduced, and the transgenic animal meansan animal into which a foreign gene is stably introduced into achromosome at an initial stage of ontogeny and the genetic character canbe transmitted to the progeny.

The transgenic animal used herein should be understood in a broad senseand includes any vertebrates other than a human being. The transgenicanimal of the present invention is useful for studies of functions orexpression control of hBSSP4 or mBSSP4, elucidation of mechanisms ofdiseases associated with cells expressing in a human being, anddevelopment of disease model animals for screening and safety test ofmedicine.

As a technique for creating the transgenic animal, a gene is introducedinto a nucleus in a pronucleus stage of egg cells with a micropipettedirectly under a phase-contrast microscope (microinjection, U.S. Pat.No. 4,873,191). Further, there are a method using embryonic stem cell(ES cell), and the like. In addition, there are newly developed methodssuch as a method wherein a gene is introduced into a retroviral vectoror adenoviral vector to infect egg cells, a sperm vector method whereina gene is introduced into egg cells through sperms, and the like.

A sperm vector method is a gene recombinant technique wherein a foreigngene is incorporated into sperm cells by adhesion, electroporation,etc., followed by fertilization of egg cells to introduce the foreigngene into the egg cells (M. Lavitranoet et al., Cell, 57, 717, 1989).Alternatively, an in vivo site specific gene recombinant technique suchas that using cre/loxP recombinase system of bacteriophage P1, FLPrecombinase system of Saccharomyces cerevisiae, etc. can be used.Furthermore, introduction of a transgene of the desired protein into anon-human animal using a retroviral vector has been reported.

For example, a method for creating a transgenic animal by microinjectioncan be carried out as follows.

First, a transgene primarily composed of a promoter responsible forexpression control, a gene encoding a specific protein and a poly Asignal is required. It is necessary to confirm expression modes andamounts between respective systems because an expression mode and amountof a specific molecule is influenced by a promoter activity, andtransgenic animals differ from each other according to a particularsystem due to the difference in a copy number of an introduced transgeneand a introduction site on a chromosome. An intron sequence which isspliced may be previously introduced before the poly A signal because ithas been found that an expression amount varies due to a non-translationregion and splicing. Purity of a gene to be used for introduction intofertilized egg cells should be as high as possible. This is ofimportance. Animals to be used include a mouse for collecting fertilizedeggs (5 to 6 week old), a male mouse for mating, a false pregnancyfemale mouse, a seminiferous tubule-ligated mouse, and the like.

For obtaining fertilized egg cells efficiently, ovulation may be inducedwith gonadotropin or the like. Fertilized egg cells are recovered and agene in an injection pipette is injected into male pronucleus of the eggcells by microinjection. For returning the injected egg cells to afallopian tube, an animal (false pregnancy female mouse, etc.) isprovided and about 10 to 15 eggs/mouse are transplanted. Then, genomicDNA is extracted from the end part of the tail to confirm whether thetransgene is introduced into newborn mouse or not. This confirmation canbe carried out by detection of the transgene with southern blottechnique or PCR technique, or by positive cloning wherein a markergene, which is activated only when homologous recombination is caused,has been introduced. Further, transcribed products derived from thetransgene are detected by northern blot technique or RT-PCR technique toconfirm expression of the transgene. Or, western blotting can be carriedout with a specific antibody to a protein.

The knockout mouse of the present invention is treated so that thefunction of mBSSP4 gene is lost. A knockout mouse means a transgenicmouse any of whose gene is destroyed by homologous recombinationtechnique so that its function is deficient. A knockout mouse can becreated by carrying out homologous recombination with ES cells andselecting embryonic stem cells wherein either of allele genes aremodified or destroyed. For example, embryonic stem cells whose genes aremanipulated at blastocyte or morula stage of fertilized eggs areinjected to obtain a chimera mouse wherein cells derived from theembryonic stem cells are mixed with those derived from the embryo. Thechimera mouse (chimera means a single individual formed by somatic cellsbased on two or more fertilized eggs) can be mated with a normal mouseto create a heterozygote mouse wherein all of either of the allele geneshave been modified or destroyed. Further, a homozygote mouse can becreated by mating heterozygote mice.

Homologous recombination means recombination between two genes whosenucleotide sequences are the same or very similar to each other in termsof gene recombination mechanism. PCR can be employed to selecthomologous recombinant cells. A PCR reaction can be carried out by usinga part of a gene to be inserted and a part of a region where theinsertion is expected as primers to find out occurrence of homologousrecombination in cells which give an amplification product. Further, forcausing homologous recombination in a gene expressed in embryonic stemcells, homologous recombinant cells can readily be selected by using aknown method or its modification. For example, cells can be selected byjoining a neomycin resistant gene to a gene to be introduced to impartneomycin resistance to cells after introduction.

The present invention also provide an antibody recognizing hBSSP4 ormBSSP4 or a fragment thereof. The antibody of the present inventionincludes an antibody against a protein having the amino acid sequencedescribed in any of SEQ ID NOS: 2, 4, 6, 18 and 20 or its fragment. Anantibody against hBSSP4 or mBSSP4 or a fragment thereof (e.g.,polyclonal antibody, monoclonal antibody, peptide antibody) or anantiserum can be produced by using hBSSP4 or mBSSP4 or a fragmentthereof, etc. as an antigen according to a per se known process forproducing an antibody or an antiserum.

The hBSSP4 or mBSSP4 or a fragment thereof is administered to a site ofa warm-blooded animal where an antibody can be produced byadministration thereof as such or together with a diluent or carrier.For enhancing the antibody production, upon administration, Freund'scomplete adjuvant or Freund's incomplete adjuvant may be administrated.Normally, the administration is carried out once every 1 to 6 weeks, 2to 10 times in all. Examples of the warm-blooded to be used includemonkey, rabbit, dog, guinea pig, mouse, rat, sheep, goat, chicken andthe like with mouse and rat being preferred. As rats, for example,Wistar and SD rats are preferred. As mice, for example, BALB/c, C57BL/6and ICR mice are preferred.

For producing monoclonal antibody producer cells, individuals whoseantibody titer have been recognized are selected from warm-bloodedanimals, e.g., a mouse immunized with an antigen. Two to 5 days afterthe last immunization, the spleen or lymph node of the immunized animalis collected and antibody producer cells contained therein are subjectedto cell fusion with myeloma cells to prepare a monoclonal antibodyproducer hybridoma. The antibody titer in an antiserum can be determinedby, for example, reacting the antiserum with a labeled hBSSP4 or mBSSP4as described hereinafter, followed by measurement of the activity boundto the antibody. The cell fusion can be carried out according to a knownmethod, for example, that described by Koehler and Milstein (Nature,256, 495, 1975) or its modifications (J. Immunol. Method, 39, 285, 1980;Eur. J. biochem, 118, 437, 1981; Nature, 285, 446, 1980). As a fusionpromoting agent, there are polyethylene glycol (PEG), Sendai virus andthe like. Preferably, PEG is used. Further, for improving fusionefficiency, lectin, poly-L-lysine or DMSO can be appropriately added.

Examples of myeloma cells include X-63Ag8, NS-1, P3U1, SP2/0, AP-1 andthe like with SP2/0 being preferred. The preferred ratio of the numberof the antibody producer cells (spleen cells):the number of spleen cellsare 1:20 to 20:1. PEG (preferably PEG 1000 to PEG 6000) is added at aconcentration of about 10 to 80% and the mixture is incubated at 20 to40° C., preferably 30 to 37° C. for 1 to 10 minutes to carry out thecell fusion efficiently. Screening of anti-hBSSP4 or mBSSP4 antibodyproducer hybridomas can be carried out by various methods. For example,a supernatant of a hybridoma culture is added to a solid phase to whichhBSSP4 or mBSSP4 antigen is adsorbed directly or together with a carrier(e.g., microplate), followed by addition of an anti-immunoglobulinantibody (in case that the cells used in cell fusion is those of amouse, anti-mouse immunoglobulin antibody is used) or protein A todetect the anti-hBSSP4 or mBSSP4 monoclonal antibody attached to thesolid phase. Or, a supernatant of a hybridoma culture is added to asolid phase to which an anti-immunoglobulin antibody or protein A isadsorbed, followed by addition of hBSSP4 or mBSSP4 labeled with aradioactive substance, an enzyme, etc., to detect the anti-hBSSP4 ormBSSP4 monoclonal antibody attached to the solid phase.

Selection and cloning of the anti-hBSSP or mBSSP monoclonal antibody canbe carried out according to a per se known method or its modification.Normally, a HAT (hypoxanthine, aminopterin, thymidine)-added medium forculturing animal cells is used. Any culture medium can be used forselection, cloning and growing up in so far as the hybridoma can grow.For example, there can be used RPMI culture medium containing 1 to 20%,preferably 10 to 20% fetal bovine serum, or a serum-free medium forculturing hybridomas. Preferably, the culture is carried out at atemperature of about 37° C. Normally, the culture time is 5 days to 3weeks, preferably 1 weeks to 2 weeks. Normally, the culture is carriedout under 5% CO₂. The antibody titer of a supernatant of a hybridomaculture can be measured according to the same manner as that of theabove-described measurement of anti-BSSP4 antibody titer in anantiserum. That is, examples of the measurement to be used includeradioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), FIA(fluorescence immunoassay), plaque assay, agglutination reaction method,and the like. Among them, ELISA as shown blew is preferred.

Screening by ELISA

A protein prepared according to the same operation as that for animmunogen is immobilized on the surface of each well of an ELISA plate.Next, BSA, MSA, OVA, KLH, gelatin, skimmed milk, or the like isimmobilized on each well to prevent non-specific adsorption. Asupernatant of a hybridoma culture is added to each well and is allowedto stand for a given time so that an immunological reaction proceeds.Each well is washed with a washing solution such as PBS or the like.Preferably, a surfactant is added to this washing solution. An enzymelabeled secondary antibody is added and allowed to stand for a giventime. As the enzyme to be used for the label, there can be usedβ-galactosidase, alkaline phosphatase, peroxidase and the like. Afterwashing each well with the same washing solution, a substrate solutionof the labeled enzyme used is added so that an enzymatic reactionproceeds. When the desired antibody is present in the supernatant of ahybridoma culture, the enzymatic reaction proceeds and the color of thesubstrate solution is changed.

Normally, cloning is carried out by a per se known method such assemi-solid agar method, limiting dilution method and the like.Specifically, after confirming a well in which the desired antibody isproduced by the above-described method, cloning is carried out to obtaina single clone. For cloning, it is preferred to employ limiting dilutionmethod wherein hybridoma cells are diluted so that one colony is formedper one well of a culture plate. For cloning by limiting dilutionmethod, feeder cells can be used, or a cell growth factor such asinterleukin 6, etc. can be added to improve colony forming capability.In addition, cloning can be carried out by using FACS and single cellmanipulation method. The cloned hybridoma is preferably cultured in aserum-free culture medium and an optimal amount of an antibody is addedto its supernatant. The single hybridoma thus obtained can be culturedin a large about by using a flask or a cell culture device, or culturedin the abdominal cavity of an animal (J. Immunol. Meth., 53, 313, 1982)to obtain a monoclonal antibody. When culturing in a flask, there can beused a cell culture medium (e.g., IMDM, DMEM, RPMI1640, etc.) containing0 to 20% of FCS. When culturing in the abdominal cavity of an animal,the animal to be used is preferably the same species or the same line asthat from which the myeloma cells used in the cell fusion are derived, athymus deficient nude mouse or the like, and the hybridoma istransplanted after administration of a mineral oil such as pristane,etc. After 1 to 2 weeks, myeloma cells are proliferated in the abdominalcavity to obtain ascites containing a monoclonal antibody.

The monoclonal antibody of the present invention which does notcross-react with other proteins can be obtained by selecting amonoclonal antibody which recognizes an epitope specific to hBSSP4 ormBSSP4. In general, an epitope presented by an amino acid sequencecomposed of at least 3, preferably 7 to 20 successive amino acidresidues in an amino acid sequence which constitutes a particularprotein is said to be an inherent epitope of the protein. Then, amonoclonal antibody recognizing an epitope constituted by a peptidehaving an amino acid sequence composed of at least 3 successive aminoacid residue selected from the amino acid residues disclosed in any ofSEQ ID NOS: 2 and 4 can be said to be the monoclonal antibody specificfor hBSSP4 or mBSSP4 of the present invention. An epitope common toBSSP4 family can be selected by selecting an amino acid sequenceconservative among the amino acid sequences described in SEQ ID NOS: 2and 4. Or, in case of a region containing an amino acid sequencespecific for each sequence, a monoclonal antibody which candifferentiate respective proteins can be selected.

Separation and purification of the anti-hBSSP4 or mBSSP4 monoclonalantibody, like a conventional polyclonal antibody, can be carried outaccording to the same manner as those of immunoglobulins. As a knownpurification method, there can be used a technique, for example, saltingout, alcohol precipitation, isoelectric precipitation, electrophoresis,ammonium sulfate precipitation, absorption and desorption with an ionexchange material (e.g., DEAE), ultrafiltration, gel filtration, orspecific purification by collecting only an antibody with anantibody-binding solid phase or an active adsorber such as protein A orprotein G, etc., and dissociating the binding to obtain the antibody.For preventing formation of aggregates during purification or decreasein the antibody titer, for example, human serum albumin is added at aconcentration of 0.05 to 2%. Alternatively, amino acids such as glycine,α-alanine, etc., in particular, basic amino acids such as lysine,arginine, histidine, etc., saccharides such as glucose, mannitol, etc.,or salts such as sodium chloride, etc. can be added. In case of IgMantibody, since it is very liable to be aggregated, it may be treatedwith β-propionilactone and acetic anhydride.

The polyclonal antibody of the present invention can be producedaccording to a per se known method or its modification. For example, animmunogen (protein antigen) per se or a complex thereof with a carrierprotein is prepared and, according to the same manner as that in theabove monoclonal antibody production, a warm-blooded animal isimmunized. A material containing an antibody against the protein of thepresent invention or its fragment is collected from the immunized animaland the antibody is separated and purified to obtain the desiredantibody. As for a complex of an immunogen and a carrier protein forimmunizing a warm-blooded animal, the kind of a carrier protein and themixing ratio of a carrier and a hapten are not specifically limited inso far as an antibody against the hapten immunized by cross-linking withthe carrier is efficiently produced. For example, there can be usedabout 0.1 to 20, preferably about 1 to 5 parts by weight of bovine serumalbumin, bovine cycloglobulin, hemocyanin, etc. coupled with one part byweight of a hapten. For coupling a carrier and a hapten, variouscondensing agents can be used. Examples thereof include glutaraldehyde,carbodiimide or maleimide active ester, active ester agents having thiolgroup or dithiopyridyl group, and the like. The condensed product isadministered as such or together with a carrier or diluent to a site ofa warm-blooded animal where an antibody can be produced. For enhancingthe antibody production, upon administration, Freund's complete adjuvantor Freund's incomplete adjuvant may be administrated. Normally, theadministration is carried out once every 2 to 6 weeks, 3 to 10 times inall. The polyclonal antibody can be collected from blood, ascites, orthe like, preferably blood of the immunized animal. The polyclonalantibody titer in an antiserum can be measured according to the samemanner as measurement of the above monoclonal antibody titer in theantiserum. Separation and purification of the polyclonal antibody, likethe above monoclonal antibody, can be carried out according to the samemanner as those of immunoglobulins.

The monoclonal antibody and polyclonal antibody against hBSSP4 or mBSSP4or a fragment thereof can be utilized for diagnosis and treatment ofdiseases associated with cells expressing hBSSP4 or mBSSP4. By usingthese antibodies, hBSSP4 or mBSSP4 or a fragment thereof can bedetermined based on their immunological binding to hBSSP4 or mBSSP4 or afragment thereof of the present invention. Specifically, examples of amethod for determining hBSSP4 or mBSSP4 or a fragment thereof in aspecimen by using these antibodies include a sandwich method wherein theantibody attached to an insoluble carrier and the labeled antibody arereacted with hBSSP4 or mBSSP4 or a fragment thereof to form a sandwichcomplex and the sandwich complex is detected, as well as a competitivemethod wherein labeled hBSSP4 or mBSSP4, and hBSSP4 or mBSSP4 or afragment thereof in the specimen are competitively reacted with theantibody and hBSSP4 or mBSSP4 or a fragment thereof in the specimen isdetermined based on the amount of the labeled antigen reacted with theantibody.

As a sandwich method for determining hBSSP4 or mBSSP4 or a fragmentthereof, there can be used two step method, one step method and thelike. In two step method, first, the immobilized antibody is reactedwith hBSSP4 or mBSSP4 or a fragment thereof and then unreacted materialsare completely removed by washing, followed by addition of the labeledantibody to form immobilized antibody-hBSSP4 or mBSSP4-labeled antibody.In one step method, the immobilized antibody, labeled antibody andhBSSP4 or mBSSP4 or a fragment thereof are added at the same time.

Examples of an insoluble carrier used for the determination includesynthetic resins such as polystyrene, polyethylene, polypropylene,polyvinyl chloride, polyester, polyacrylate, nylon, polyacetal, fluorineplastic, etc.; polysaccharides such as cellulose, agarose, etc.; glass;metal; and the like. An insoluble carrier may be shaped in variousforms, for example, tray, sphere, fiber, rod plate, container, cell,test tube, and the like. The antibody adsorbed by a carrier is stored ata cold place in the presence of an appropriate preservative such assodium azide or the like.

For immobilization of the antibody, a known chemical bonding method or aphysical adsorption can be used. Examples of a chemical bonding methodinclude a method using glutaraldehyde; maleimide method usingN-succusinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,N-succusinimidyl-2-maleimide acetate or the like; carbodiimide methodusing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; orthe like. In addition, there are maleimidobenzoyl-N-hydroxysuccinimideester method, N-succinimidyl-3-(2-pyridylthio)propionic acid method,bisdiazobenzidine method, and dipalmityllysine method. Or, it ispossible to capture a complex formed beforehand by reacting a materielto be tested with two antibodies, whose epitopes are different, with animmobilized a 3rd antibody against the antibody.

For labeling, it is preferred to use enzyme, fluorescent substance,luminous substance, radioactive substance, metal chelate, or the like.Examples of the enzyme include peroxidase, alkaline phosphatase,β-D-galactosidase, malate dehydrogenase, Staphylococcus nuclease,δ-5-steroidisomerase, α-glycerol phosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, asparaginase, glucoseoxidase, ribonuclease, urease, catalase, glucose-6-phosphatedehydrogenase, glucoamylase, acetylcholinesterase and the like. Examplesof the fluorescent substance include fluorescein isothiocyanate,phycobiliprotein, rhodamine, phycoerythrin, phycocyanin,allophycocyanin, o-phthalaldehyde, and the like. Examples of theluminous substance include isoluminol, lucigenin, luminol, aromaticacridinium ester, imidazole, acrdinium salt and its modified ester,luciferin, luciferase, aequorin and the like. Examples of theradioactive substance include ¹²⁵I, ¹²⁷I, ¹³¹I, ¹⁴C, ³H, ³²P, ³⁵S andthe like. The labeling material is not limited to them and any materialwhich can be used for immunological determination can be used. Further,a low molecular weight hapten such as biotin, dinitrophenyl, pyridoxalor fluorescamine may be attached to the antibody. Preferably,horseradish peroxidase is used as a labeling enzyme. This enzyme can bereacted with various substrates and can readily be attached to theantibody by periodate method.

When an enzyme is used as a labeling material, a substrate and, ifnecessary, a coloring enzyme is used for measuring its activity. In caseof using peroxidase as the enzyme, H₂O₂ is used as a substrate and, as acoloring agent, there can be used 2,2′-azino-di-[3-ethylbenzthiazolinesulfonic acid] ammonium salt (ABTS), 5′-aminosalicylic acid,o-phenylenediamine, 4-aminoantipyrine, 3,3′,5,5′-tetramethylbenzidineand the like. In case of using alkaline phosphatase as the enzyme,o-nitorphenylphosphate, p-nitrophenylphosphoric acid, or the like can beused as a substrate. In case of using β-D-galactosidase as the enzyme,fluorescein-d-(β-D-galactopyranoside),4-methylumbelliphenyl-β-D-galactopyranoside, or the like can be used asa substrate. The present invention also include a kit comprising theabove monoclonal antibody, polyclonal antibody and reagents.

As a cross-linking agent, a known cross-linking agent such asN,N′-o-phenylenedimaleimide,4-(N-maleimidomethyl)cyclohexanoate-N-succinimide ester,6-maleimidohexanoate-N-succineimide ester, 4,4′-dithiopyridine or thelike can be utilized. The reaction of these cross-linking agents withenzymes and antibodies can be carried out by a known method according toproperties of a particular cross-linking agent. Further, as theantibody, a fragment thereof, for example, Fab′, Fab, F(b′2) can be usedas the case may be. A labeled enzyme can be obtained by the sametreatment regardless of whether the antibody is polyclonal ormonoclonal. When the above labeled enzyme obtained by using across-linking agent is purified by a known method such as affinitychromatography or the like, a immunoassay system having more highersensitivity can be obtained. The enzyme labeled and purified antibody isstored at a dark cold place with addition of a stabilizer such asthimerosal, glycerin or after lyophilization.

An objective to be determined is not specifically limited in so far asit is a sample containing BSSP4 or a fragment thereof, or a samplecontaining a precursor of BSSP4 or a fragment thereof and includes bodyfluids such as plasma, serum, blood, serum, urine, tissue fluid,cerebrospinal fluid and the like.

The following Examples further illustrate the present invention indetail but are not construed to limit the scope thereof.

EXAMPLE 1

Cloning of Novel Serine Protease mBSSP4 Gene

The cloning was carried out by PCR using a human brain cDNA library(Clontech) as a template and nucleotide sequences corresponding to anamino acid sequence common to serine proteases represented by Primer 1:GTG CTC ACN GCN GCB CAY TG (SEQ ID NO: 30) Primer 2: CCV CTR WSD CCN CCNGGC GA (SEQ ID NO: 31)as primers. Namely, 5 μl of the template, 5 μl of 10×ExTaq buffer, 5 μlof DNTP, 10 pmol of each of the above primers and 0.5 μl of ExTaq(TAKARA) were added and the total volume was adjusted to 50 μl withsterilized water. PCR was carried out by repeating a cycle of heating at94° C. for 0.5 minute, at 55° C. for 0.5 minute and then at 72° C. for 1minutes, 35 times. The PCR product was mixed with pCR II-TOPO vectorattached to TOPO TA cloning kit (Invitrogen) and the mixture was allowedto stand at room temperature for 5 minutes. Then, according to aconventional manner, E. coli Top 10 attached to the kit was transformedand applied to a LB (Amp+) plate (containing 100 μg/ml of ampicillin).According to a conventional manner, a plasmid was extracted from eachcolony obtained and its nucleotide sequence was determined by cyclesequencing method with a fluorescence sequencer (ABI). Homology of thesequence of each clone was examined by means of GenBank. Regarding anunknown sequence, i.e., BSSP4 gene, the full length cDNA was obtained by5′ RACE and 3′ RACE and, according to the same manner as describedabove, the nucleotide sequence was determined. Namely, BSSP4 clonespecific primers, GSP1 primers [hBSSP4F1 (SEQ ID NO: 32) or hBSSP4R1(SEQ ID NO: 36)] and GSP2 primers [hBSSP4F2 (SEQ ID NO: 33) or hBSSP4R2(SEQ ID NO: 37)] were prepared. PCR was carried out by using human brainMarathon-Ready cDNA (Clontech), AP1 primer attached to this reagent andeither of the above GSP1 primers and heating at 94° C. for 2 minutesonce and repeating a cycle of heating at 94° C. for 30 seconds, at 60°C. for 30 seconds and then at 72° C. for 30 seconds 35 times. Then, 5 μlof the PCR product diluted to 1/100, 5 μl of 10×buffer, 5 μl of dNTP, 10pmol of either of 10 μM of the above GSP2 primer, 10 pmol of AP2 primerattached to the above reagent and 0.5 unit of ExTaq were admixed andadjusted to 50 μl with sterilized water. Then, according to the samemanner as the above, PCR was carried out. The PCR product was cloned bythe above TOPO TA cloning kit and sequenced to obtain the upstream anddownstream regions of the above clone. At this time, as for a clonewhich seemed not to cover the full length of a protein, the specificprimers shown hereinafter were prepared based on the newly foundnucleotide sequence. Further, based on this sequence, the primerscapable of amplifying ORF as shown hereinafter [hBSSP4F6 (SEQ ID NO: 35)and hBSSP4R3/E (SEQ ID NO: 38) or hBSSP4R4/E (SEQ ID NO: 39)] wereprepared and PCR carried out using human brain Marathon-ready cDNA as atemplate to confirm that these clones were identical. This was clonedinto pCR II-TOPO vector attached to TOPO TA cloning kit to obtain theplasmid pCR II/hBSSP4 containing the full length cDNA clone. Thenucleotide sequence of DNA contained in this plasmid is shown in SEQ IDNO: 1 and the amino acid sequence of hBSSP4 protein deduced from thenucleotide sequence is shown in SEQ ID NO: 2. Further, two differenttypes of clones were obtained. The amino acid sequence of hBSSP4represented by SEQ ID NO: 2 (the 1st to 268th amino acids) is hBSSP4mature or active type protein composed of 268 amino acids. In the aminoacid sequence represented by SEQ ID NO: 2, the −49th to −1st amino acidsare a prepro or pro part and the −15th to −1st amino acids are a propart and are considered to be a precursor of hBSSP4. As consensussequences of serine proteases, there are Ala-Ala-His-Cys represented bythe 39th to 42nd amino acid residues of SEQ ID NO:2 andAsp-Ser-Gly-Gly-Pro represented by the 192nd to 196th amino acidresidues of SEQ ID NO:2 and there are one or more Asp's between theseconsensus sequences.

Further, 8 clones having different nucleotide sequences, perhaps, causedby alternative splicing wre obtained. The nucleotide sequences thereofare shown in SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15 and 17. Further, theamino acid sequeces thereof deduced from these nucleotide sequences areshown in SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16 and 18. As described above,in these sequences, there are those having either or bot consensussequences of serine proteases and those having no consensus sequences ofserine proteases. There is a possibility that these gene products(transcription product or translation product) have a function ofcontrol factors for serine proteases.

According to the same manner, 5′ RACE and 3′ RACE were carried out byusing the primers as described hereinafter and mouse brainMarathon-Ready cDNA (Clontech) as a template, followed by cloning toobtain mouse homologous gene pCRII/mBSSP4. The nucleotide of DNAcontaining this plasmid is shown by SEQ ID NO:19 and the amino acidsequence of mBSSP4 protein deduced from this nucleotide sequence isshown in SEQ ID NO:20. The amino acid sequence of mBSSP4 represented bySEQ ID NO:20 (the 1st to 259th amino acids) is mBSSP4 mature or activetype protein composed of 259 amino acids. In the amino acid sequencerepresented by SEQ ID NO:20, the −49th to 1st amino acids are a preproor pro part and the −15th to −1st amino acids are a pro part and areconsidered to be a precursor of mBSSP4. As consensus sequences of serineproteases, there are Ala-Ala-His-Cys (the 39th to 42nd amino acidresidues of SEQ ID NO:20) and Asp-Ser-Gly-Gly-Pro (the 192nd to 196thamino acid residues of SEQ ID NO:20) and there are one or more Asp'sbetween the consensus sequences. human BSSP4 hBSSP4F1 ForwardAGGTTCCTATCATCGACTCG RACE (SEQ ID NO: 32) hBSSP4F2 ForwardTGAGGACATGCTGTGTGCCGG RACE (SEQ ID NO: 33) hBSSP4F3 ForwardGTTGTGGGCGGCGAGGACAG mature (SEQ ID NO: 34) hBSSP4F6 ForwardGCCATGGTGGTTTCTGGAGC FL* (SEQ ID NO: 35) hBSSP4R1 ReverseTATGGTTTGTTCAGGTTGTCC RACE (SEQ ID NO: 36) hBSSP4R2 ReverseAGGGCAATGTCTGCACAGGC RACE (SEQ ID NO: 37) hBSSP4R3/E ReverseCTGAATTCCTAGGAGCGCGCGGCGGCC FL* (SEQ ID NO: 38) hBSSP4R4/E ReverseGAGAATTCGATATGTGGGCAGGGTTACA FL* (SEQ ID NO: 39) mouse BSSR4 mBSSP4.1Forward ACAAACCATCTCTGTTCTCAG RACE (SEQ ID NO: 40) mBSSP4F2 ForwardGTCCCAGAAAGTAGGCATTG RACE (SEQ ID NO: 41) mBSSP4F3 ForwardCTCCACCCATACCAGCAATG FL* (SEQ ID NO: 42) mBSSP4F4 ForwardATTGTGGGAGGTGAGGACAG mature (SEQ ID NO: 43) mBSSP4.2 ReverseTGCAGAGTTCGGAGTCGATG RACE (SEQ ID NO: 44) mBSSP4R2 ReverseATCCAGCAGTCGGTCTTGGG RACE (SEQ ID NO: 45) mBSSP4R3/P ReverseATTCTGCAGTTCCTTGTTCTCTCGCTCAGGFL* (SEQ ID NO: 46)*: for full length

EXAMPLE 2

Expression hBSSP4 or mBSSP4 Gene in Human Being and Mouse InternalOrgans

According to the protocol of QuickPrep Micro mRNA purification Kit(Amersham-Pharmacia), mRNAs were isolated from various internal organsof Balb/c mice or their fetuses. They were subjected to electrophoresisaccording to a conventional manner and transcribed to a nylon membrane.A probe was prepared separately by isolating a part of a nucleotidesequence encoding the mature protein of mBSSP4 from pCR II/mBSSP4,purifying it and labeling it with α-³²p dCTP. The probe was diluted with5×SSC and reacted with the above membrane filter at 65° C. for a wholeday and night. Likewise, a probe was prepared by isolating a part of anucleotide sequence encoding the mature protein of hBSSP4 from pCRII/hBSSP4, purifying it and labeling it with α-³²P dCTP, and dilutedwith 5×SSC and the dilution was reacted with human multiple tissue blot,human multiple tissue blot II and human brain multiple blot II(Clontech) membrane. Then, the filter was washed twice each with2×SSC/0.1% SDS at room temperature for 30 minutes, 1×SSC/0.1% SDS atroom temperature for 30 minutes and 0.1×SSC/0.1% SDS at 65° C. for 30minutes. The filter was exposed to an imaging plate for FLA2000 (FujiFilm) for one day to analyze the expression. The results shown in thedrawings are those obtained by using human multiple tissue blot membrane(FIG. 1), human multiple tissue blot II membrane (FIG. 2), human brainmultiple blot II membrane (FIG. 3) and mRNAs prepared from variousinternal organs of 3-month-old mice (FIG. 4) and mRNAs prepared fromprostates of 1-month-old, 3-month-old and 12-month-old mice (FIG. 5). Inaddition, the mRNAs prepared above were subjected to RT-PCR by usingReady To Go RT-PCR Beads (Amersham-Pharmacia) and hBSSP4 and mBSSP4 genespecific primers (human being: SEQ ID NOS: 33 and 38 or 39, mouse: SEQID NOS: 40 and 44) according to the protocol attached to the kit.

As seen form FIGS. 1 to 3, in case of northern blotting analysis, theexpression of hBSSP4 was recognized in prostate (FIG. 2, the bandbetween 1.35 to 2.4 kb) and cerebllum (FIG. 3, the band between 1.35 and2.4 kb). The expression of mBSSP4 was recognized in prostate andskeletal muscle (FIG. 4). Further, according to the results of RT-PCR,the expression of hBSSP4 was recognized in brain, placenta, testicle andprostate of from fetuses to adults of human beings and the expression ofmBSSP4 was recognized in prostate of newborns to adults of mice. Then,it is considered that the novle serine proteases of the presentinvention have various roles in brain, prostate, placenta, testicle andskeletal muscle. Further, the presence of the transcribed product (about1.4 to 1.5 kb) having the nucleotide sequence of SEQ ID NO: 7 has beenconfirmed by the above northern blotting analysis.

EXAMPLE 3

Expression of Novel Serine Proteases Encoded by hBSSP4 or mBSSP4 Gene

(1) Construction of Expression Plasmid

A cDNA region encoding the mature form of hBSSP4 or mBSSP4 protein wasamplified by PCR using the plasmid pCR II/hBSSP4 or pCR II/mBSSP4 as atemplate (the primers used were those having the sequences of SEQ IDNOS: 34 and 39 for a human being and those having the sequences of SEQID NOS: 43 and 46 for mouse). The PCR product was ligated to pTrc-HisB(Invitrogen) which had been digested with BamHI and blunted with mungbean nuclease. E. coli JM109 was transformed by the resultant andcolonies formed were analyzed by PCR to obtain E. coli containing thedesired serine protease expressing plasmid pTrcHis/hBSSP4 orpTrcHis/mBSSP4.

The resultant E. coli strains were designated E. coli pTrcHis/hBSSP4 andE. coli pTrcHis/mBSSP4, respectively, and deposited at NationalInstitute of Bioscience and Human-Technology (NIBH), Agency ofIndustrial Science & Technology of 1-1-3 Higashi, Tsukuba-shi,Ibaraki-ken, Japan on Oct. 29, 1998 under the accession numbers of FERMP-17037 and FERM P-17034, respectively.

(2) Expression of Protein by E. coli Containing Expression Plasmid

A single colony of E. coli having the expression plasmid was inoculatedin 10 ml of LB (Amp⁺) culture medium and incubated at 37° C. overnight.This was inoculated in 250 ml of LB (Amp⁺) culture medium and incubatedat 37° C. When the absorbance at 600 nm became 0.5, 250 μl of 0.1 M IPTG(isopropyl-β-D-(−)-thiogalactopyranoside) was added and the incubationwas continued for additional 5 hours. The E. coli was centrifuged andsuspended in a cell disruption buffer (10 mM phosphate buffer pH 7.5, 1mM EDTA) and sonicated on ice to disrupt E. coli. This was centrifugedat 14,000 r.p.m. for 20 minutes to obtain a precipitate. The precipitatewas washed twice with a cell disruption buffer containing 0.5% TritonX-100™ and washed with water to remove Triton X-100™. Then, theresultant mixture was dissolved by soaking in a denaturation buffercontaining 8 M urea (8M urea, 50 mM Tris pH8.5, 20 mM ME) at 37° C. for1 hour. The solution was passed through TALON metal affinity resin(Clontech), washed with the denaturation buffer containing 10 mMimidazole, and then eluted with the denaturation buffer containing 100mM imidazole to purify the solution. The purified product was dialyzedagainst PBS for 3 days with exchanging the buffer every other night toobtain the protein hBSSP4-His or mBSSP4-His.

Example 4

Expression of Novel Serine Protease Mature Protein Encoded by hBSSP4Gene by Using pFBTrypSigTag/hBSSP4

(1) Construction of pFBTrypSigTag/hBSSP4

The sequences represented by SEQ ID NOS: 21 and 22 were subjected toannealing and digested with NheI and BamHI. The resultant fragment wasinserted into Nhe-I-BamHI digested pSecTag2A (Invitrogen) to obtainpSecTrypHis. Twenty units of BamHI was added to 5 μg of pSecTrypHisvector and the vector was cleaved at 37° C. over 4 hours. Then, 6 unitsof mung bean nuclease (TAKARA) was added thereto and reacted at roomtemperature (25° C.) for 30 minutes to blunt the terminal ends. Further,the 3′-terminus side of the cloning site was cleaved with 20 units ofXhoI, 1 unit of bacterial alkaline phosphatase (TAKARA) was addedthereto and the reaction was carried out at 65° C. for 30 minutes.

According to the same manner as that described in JP 9-149790 A orBiochim. Biophys. Acta, 1350, 11, 1997, mRNA was prepared from COLO201cells and cDNA was synthesized to obtain the plasmid pSPORT/neurosin.cDNA of an active region of neurosin was obtained from pSPORT/neurosinby PCR using primers having the sequences represented by SEQ ID NOS: 23and 24. Ten units of XhoI was reacted with the PCR product at 37° C. for3 hours to cleave XhoI site at the 3′-side thereof. This was insertedinto pSecTrypHis by TAKARA ligation kit to obtain pSecTrypHis/neursoin(FIG. 6).

Amplification was carried out by using the primers having the sequencesrepresented by SEQ ID NOS: 25 and 26 so that the peptide ofLeu-Val-His-Gly SEQ ID NO:52 was present at the C-terminus of the partfrom trypsin signal to the enterokinase recognition site ofpSecTrypHis/neurosin. This was inserted between NheI and HindIII sitesof pSecTag2A to construct the plasmid pTrypSig.

One μg (0.1 μl) of the plasmid pSecTab2A was treated with therestriction enzymes NheI and BamHI to completely remove a regionencoding the leader sequence of IgGk. One hundred pmol portions of DANsrepresented by SEQ ID NOS: 47 and 48 were added to the resultantsolution and the mixture was heated at 70° C. for 10 minutes andsubjected to annealing by allowing to stand at room temperature for 30minutes. Two μl of I solution of DNA ligation kit Ver. 2 (TAKARA) wasadded to 1 μl portions of His secretory signal sequence and pSecTag2Atreated by NheI and BamHI and the reaction was carried out at 16° C. for30 minutes.

To the reaction mixture was add 0.1 ml of E. coli competent cellXL1-Blue (STRATAGENE) and reacted on ice for 30 minutes. Then, thereaction mixture was subjected to heat shock at 42° C. for 60 seconds.After standing on ice for 2 minutes, 0.9 ml of SOC culture medium (ToyoBoseki K. K.) was added thereto and the mixture was shaken with a shakerat 37° C. for 1 hour. The mixture was centrifuged at 5,000 r.p.m. for 1minutes and the supernatant was discarded. The precipitated competentcells were suspended in the liquid remained in the centrifuge tube andthe suspension was applied to an ampicillin LB plates containing 100μg/ml of ampicillin. The plates were incubated at 37° C. overnight.Among the colonies formed, a colony into which DNA of His secretorysignal was inserted was selected by PCR to obtain pTrypHis.

A sequence of about 200 bp containing His Tag region of pTrypHis wasamplified by using primers having the sequence represented by SEQ IDNOS: 26 and 27 and a fragment of about 40 bp containing His Tag andenterokinase recognizing site formed by digestion of HindIII and BamHIwas inserted into pTrypSig to construct pTrypSigTag (FIG. 7A).

cDNA was prepared by PCR of the sequence from trypsin signal toenterokinase recognizing site of pTrypSigTag using primers having thesequences represented by SEQ ID NOS: 24 and 28 and cut out by digestionwith BglII and BamHI. It was inserted into BamHI site of pFastBAC1(GIBCO). The insertion direction was confirmed by PCR using primershaving the sequences represented by SEQ ID NOS 24 and 29. A clone intowhich the cDNA was inserted in the direction toward transcription andtranslation by polyhedrin promoter was selected to obtain pFBTrypSigTag.

Twenty units of BamHI was added to 5 μg of pFBTrypSigTag vector and thevector was cleaved at 37° C. over 4 hours, followed by addition of 6units of mung bean nuclease (TAKARA) and reaction at room temperature(25° C.) for 30 minutes to blunt the terminal ends. Further, the 3′-sideof the cloning site was cleaved by 20 units of EcoRI, followed byaddition of 1 unit of bacterial alkaline phosphatase (TAKARA). Thereaction was carried out at 65° C. for 30 minutes.

cDNA of the active region of hBSSP4 was obtained by PCR according to aconventional manner using pTrcHis/hBSSP4 prepared from E. colipTrcHis/hBSSP4 (accession No. FERM P-17037) or pCRII/hBSSP4. Theresultant cDNA was inserted into pFBTrypSigTag to obtainpFBTrypSigTag/hBSSP4 (FIG. 7B). At this time, correct insertion ofhBSSP4 was confirmed by determining the sequence.

Bacmid DNA was transformed with pFBTrypSigTag/hBSSP4 according to aprotocol of Gibco BRL BAC-TO-BAC baculovirus expression system toprepare a recombinant bacmid having chimera hBSSP4 fused withtrypsinogen signal peptide, His tag and enterokinase recognizing site.When this was expressed in Sf-9 cell according to a manual of BAC-TO-BACbaculovirus expression system, it was secreted in the culturesupernatant from 2 days after infection of the virus.

According to the same manner as described above, pFETrypSigTag/mBSSP4can be prepared and secreted by using pTrcHis/mBSSP4 obtained from E.coli pTricHis/mBSSP4 (accession No. FERM P-17034) or pCRII/mBSSP4obtained in Example 1.

(2) Determination of Enzyme Activity

The recombinant fused protein hBSSP4 obtained in the culture supernatantwas passed through a chelate column to purify it and, after dialysis,its enzyme activity was determined. First, the culture supernatant wasapplied to a chelate column (Ni-NTA-Agarose, Qiagen) with PBS buffer andeluted stepwise with a solution of imidazole (Wako Pure ChemicalIndustries, Ltd.) dissolved in PBS. The resultant imidazole-elutedfraction was applied to a PD-10 column (Pharmacia) to exchange to PBSbuffer. Fifty μl of this sample was mixed with 10 μl of enterokinase (1U/1 μl, Invitrogen) and the reaction was carried out at room temperaturefor 60 minutes. Each of various synthetic substrates (PeptideLaboratory, Boc-Gln-Ala-Arg-MCA, Boc-Phe-Ser-Arg-MCA, Bz-Arg-MCA,Boc-Val-Leu-Lys-MCA, Pyr-Gly-Arg-MCA, Pro-Phe-Arg-MCA,Boc-Val-Pro-Arg-MCA, Z-Arg-Arg-MCA, Arg-MCA, Z-Phe-Arg-MCA) wasdissolved in DMSO and diluted with 1 M Tris-HCl (pH 8.0) to obtain asubstrate solution. Fifty μl of 0.2 M substrate solution was addedthereto and further the reaction was carried out at 37° C. After onehour, the fluorescence of AMC (7-amino-4-methylcoumalin) formed by theenzymatic reaction was measured at 380 nm of excitation wavelength and460 nm of fluorescence wavelength to determine the activity.

As a result, the recombinant fused protein hBSSP4 has been shown to haveserine protease activity. Likewise, mBSSP4 derived from a mouse showedthe activity.

Industrial Utililty

According to the present invention, there are provided isolated humanand mouse serine protease (hBSSP4 and mBSSP4) polynucleotides, theirhomologous forms, mature forms, precursors and polymorphic variants.Further, according to the present invention, there are provided hBSSP4and mBSSP4 proteins as well as compositions containing hBSSP4 and mBSSP4polynucleotides and proteins, their production and use.

Sequence Listing Free Text

SEQ ID NO: 21: Designed oligonucleotide to construct plasmid pSecTrypHis

SEQ ID NO: 22: Designed oligonucleotide to construct plasmid pSecTrypHis

SEQ ID NO: 23: Designed oligonucleotide primer to amplifyneurosin-encoding sequence

SEQ ID NO: 24: Designed oligonucleotide primer to amplifyneurosin-encoding sequence

SEQ ID NO: 25: Designed oligonucleotide primer to amplify a portion ofplasmid pSecTrypHis/Neurosin

SEQ ID NO: 26: Designed oligonucleotide primer to amplify a portion ofplasmid pSecTrypHis/Neurosin

SEQ ID NO: 27: Designed oligonucleotide primer to amplify a portion ofplasmid pTrypHis

SEQ ID NO: 28: Designed oligonucleotide primer to amplify a portion ofplasmid pTrypSigTag

SEQ ID NO: 29: Designed oligonucleotide primer to amplify a portion ofplasmid pFBTrypSigTag

SEQ ID NO: 30: Designed oligonucleotide primer to amplify conservedregion of serin proteases-encoding sequence; n is a, c, g or t.

SEQ ID NO: 31: Designed oligonucleotide primer to amplify conservedregion of serin proteases-encoding sequence; n is a, c, g or t.

SEQ ID NO: 32: Designed oligonucleotide primer designated as hBSSP4F1for RACE for human BSSP4 (forward)

SEQ ID NO: 33: Designed oligonucleotide primer designated as hBSSP4F2for RACE for human BSSP4 (forward)

SEQ ID NO: 34: Designed oligonucleotide primer designated as hBSSP4F3 toamplify mature human BSSP4-encoding region (forward)

SEQ ID NO: 35: Designed oligonucleotide primer designated as hBSSP4F6 toamplify full-length human BSSP4-encoding mRNA (forward)

SEQ ID NO: 36: Designed oligonucleotide primer designated as hBSSP4R1for RACE for human BSSP4 (reverse)

SEQ ID NO: 37: Designed oligonucleotide primer designated as hBSSP4R2for RACE for human BSSP4 (reverse)

SEQ ID NO: 38: Designed oligonucleotide primer designated as hBSSP4R3/Eto amplify full-length human BSSP4-encoding mRNA (reverse)

SEQ ID NO: 39: Designed oligonucleotide primer designated as hBSSP4R4/Eto amplify full-length human BSSP4-encoding mRNA (reverse)

SEQ ID NO: 40: Designed oligonucleotide primer designated as mBSSP4.1for RACE for mouse BSSP4 (forward)

SEQ ID NO: 41: Designed oligonucleotide primer designated as mBSSP4F2for RACE for mouse BSSP4 (forward)

SEQ ID NO: 42: Designed oligonucleotide primer designated as mBSSP4F3 toamplify full-length mouse BSSP4-encoding mRNA (forward)

SEQ ID NO: 43: Designed oligonucleotide primer designated as mBSSP4F4 toamplify mature mouse BSSP4-encoding region (forward)

SEQ ID NO: 44: Designed oligonucleotide primer designated as mBSSP4.2for RACE for mouse BSSP4 (reverse)

SEQ ID NO: 45: Designed oligonucleotide primer designated as mBSSP4R2for RACE for mouse BSSP4 (reverse)

SEQ ID NO: 46: Designed oligonucleotide primer designated as mBSSP4R3/Pto amplify full-length mouse BSSP4-encoding mRNA (reverse)

SEQ ID NO: 47: Designed oligonucleotide to construct plasmid pTrypHis

SEQ ID NO: 48: Designed oligonucleotide to construct plasmid pTrypHis

1. A protein selected from the group consisting of: (a) a protein havingthe amino acid sequence composed of 270 amino acids represented by the1^(st) to 270^(th) amino acids of SEQ ID NO:4; (b) a protein having theamino acid sequence composed of 319 amino acids represented by the−49^(th) to 270^(th) amino acids of SEQ ID NO:4; and (c) a proteinhaving the amino acid sequence composed of 285 amino acids representedby the −15^(th) to 270^(th) amino acids of SEQ ID NO:4.
 2. A nucleotidesequence selected from the group consisting of: (i) a nucleotidesequence represented by the 151^(st) to 960^(th) bases of SEQ ID NO:3;(ii) a nucleotide sequence encoding the amino acid sequence representedby the 1^(st) to 270^(th) amino acids of SEQ ID NO:4; (III) a nucleotidesequence hybridizable with a nucleotide sequence which is complementaryto the above nucleotide sequence (i) or (ii) under stringent conditionsof incubation in a solution containing 5×SSC or SSPE, 5% Denhardt'ssolution (01% BSA, 0.1% Ficol 1400, 0.1% PVP), 0.5% SDS and 20 μg/mldenatured salmon sperm DNA 37° C. overnight, followed by washing with2×SSC or SSPE containing 0.1% SDS at room temperature, and encoding aprotein having the same serine protease property as that of the proteinhaving the amino acid sequence represented by the 1^(st) to 270^(th)amino acids of SEQ ID NO:4; (iv) a nucleotide sequence represented bythe 4^(th) to 960^(th) bases of SEQ ID NO:3; (v) a nucleotide sequenceencoding the amino acid sequence represented by the −49^(th) to 270^(th)amino acids of SEQ ID NO:4; (vi) a nucleotide sequence hybridizable witha nucleotide sequence which is complementary to the above nucleotidesequence (iv) or (v) under stringent cnditoins of incubation in asolution containing 5×SSC or SSPE, 5% Denhardt's solution (0.1% BSA,0.1% Ficol 1400, 0.1% PVP), 0.5% SDS and 20 μg/ml denatured salmon spermDNA at 37° C. overnight, followed by washing with 2×SSC or SSPEcontaining 0.1% SDS at room temperature, and encoding a protein havingthe same serine protease property as that of the preotein having theamino acid sequence represented by the −49^(th) to 270^(th) amino acidsof SEQ IDN O:4; (vii) a nucleotide sequence represented by the 106^(th)to 960^(th) bases of SEQ ID NO:3; (viii) a nucleotide sequence encodingthe amino acid sequence represented by the −15^(th) to 270^(th) aminoacids of SEQ ID NO:4; (ix) a nucleotide sequence hybridizable with anucleotide sequence which is complementary to the above nucleotidesequence (vii) or (viii) under stringent conditions of incubation in asolution containing 5×SSC or SSPE, 5% Denhardt's solution (0.1% BSA,0.1% Ficol 1400, 0.1% PVP), 0.5% SDS and 20 μg/ml denatured salmon spermDNA at 37° C. overnight, followed by washing with 2×SSC or SSPEcontaining 0.1% SDS at room temperature, and encoding a protein havingthe same serine protease property as that of the protein encoded by thenucleotide sequence represented by SEQ ID NO:3.
 3. A vector, comprisingthe nucleotide sequence according to claim
 2. 4. Transformed cellshaving the nucleotide sequence according to claim 2 in an expressiblestate.
 5. A process for producing a protein which comprises culturingcells transformed with the nucleotide sequence according to (1) to (xi)of claim 2 or a fragment thereof, and collecting hBSSP4 produced.
 6. Theprocess according to claim 5, wherein the cells are E. coli cells,animal cells or insect cells.
 7. A method for screening for an inhibitorof serine protease, comprising comparing the enzyme activity of theprotein according to claim 1 upon bringing it into contact with acandidate compound with the enzyme activity of the protein withoutcontact with the candidate compound.
 8. A pharmaceutical composition,comprising the protein according to claim 1.